Photocaged Fluorogenic Glycoprobes for Light-Activatable Sensing and Imaging of Galactosidase Activities in Live Cells

The accurate sensing and imaging of enzymatic activities in live cells is key to exploring their intrinsic biological and pathological functions. While previously developed molecular probes are directly activated in the presence of a target enzyme, here, we developed a photocaging strategy for enzymatic substrates to enable tandem light-activatable, fluorogenic enzyme recognition as an approach with improved sensing fidelity. Galactosidases were used as the target enzyme in a proof-of-concept approach. We synthesized α- and β-galactose-conjugated, rhodol-based glycoprobes, in which the C6-position of the galactosyl substrates was modified with a photolabile protecting group, ortho-nitrobenzyl alcohol (o-NBA). We demonstrated that the presence of a selective galactosidase did not cause the fluorescence of the corresponding photocaged glycoprobe to enhance, whereas pretreatment with light irradiation to remove the photocage gave rise to fluorescence enhancement due to exposure of the natural galactosyl substrates to target enzymes. We also demonstrated the applicability of the photocaged glycoprobes for light-activatable endogenous galactosidase imaging in live cells, thus highlighting the potential of using our photocaging strategy for light-activatable, on-demand release of a fluorogenic probe capable of precisely monitoring enzymatic activities in complex biological systems.